Hyperhomocysteinemia is a strong independent risk factor for cardiovascular disease and is an emerging risk factor for Alzheimer's disease. The focus of our work has been on: 1) homocysteine metabolism in human vascular cells and tissues, 2) biochemistry of homocysteine in the circulation, and 3) pathogenic mechanisms of homocysteine in atherogenesis and the progression of vascular disease. We hypothesized that vascular cells would be particularly vulnerable to the increased levels of homocysteine that occur in hyperhomocysteinemia because of their limited capacity to metabolize homocysteine. We also suggest that homocysteine pathogenicity is not due to its ability to generate reactive oxygen species, hence oxidative stress, when undergoing oxidation in the circulation. We believe that homocysteine attacks specific molecular targets and adversely modulates their biological activity. We call this the "molecular target hypothesis", which we offer as an alternative to the "oxidative stress hypothesis". The long-term objectives of this work are: 1) to identify intracellular and extracellular molecular targets of homocysteine and understand how homocysteine interacts and modulates their activity, 2) to learn how homocysteine-targeted molecules cause cellular dysfunction, and 3) with this knowledge, develop strategies to lower tHcy in disease states such as renal failure. Our specific aims are: 1) to study the formation and biochemistry of the circulating forms of homocysteine. This aim is driven by the hypothesis that all forms of homocysteine in circulation play important roles in atherogenesis and other disease processes, 2) to study homocysteine transport, metabolism and gene-nutrient interactions in vascular cells. This aim is driven by the hypothesis that folate/cobalamin-dependent remethylation is the only pathway available in vascular cells for homocysteine metabolism and that the efficiency of remethylation is dependent upon cofactor availability and enzyme functionality, and 3) to study the effect of homocysteine on endothelial and smooth muscle cell function. This aim is driven by the hypothesis that homocysteine alters vascular cell function by targeting and modulating the activity of specific molecules on and within the cell.